FIGURE 4.

Signaling pathways associated with the protective effect of metformin on endothelial senescence in HUVECs. HUVECs were treated for 24 h with 0.5 µM DOX ± metformin (2 and 5 mM, added 24 h before DOX). Thereafter, DOX was removed and the cells were incubated in DOX-free media with or without metformin for an additional 72 h. Protein expressions of phospho- (A) JNK, (B) p38, (C) NF-κB p65, and (D) AMPK (n = 4–7) were quantified using western blot. Representative images of western blots are shown. Values were normalized to total protein or α-tubulin and expressed relative to cells treated with DOX alone. Expressed values are presented as mean ± SEM. Data were analyzed by one-way ANOVA followed by a Dunnet’s multiple comparisons test (Figure 4A,C) or non-parametric Kruskal–Wallis tests followed by Dunn’s post hoc test (Figure 4B,D); * p <0.05, *** p <0.001, **** p < 0.0001.